Epigenetic modifications, which result in heritable changes in gene function that do not involve changes to the DNA sequence, play central roles in human brain development. Dynamic regulation of DNA methylation by de novo DNA methyltransferases (Dnmts) is implicated in the control of neuronal and glial cell differentiation. We recently discovered that the Dnmt3a gene is a direct thyroid hormone (TH) receptor (TR) target in mouse brain. Postembryonic brain development is critically dependent on thyroid hormone (TH); TH deficiency during fetal and neonatal periods of human development leads to a condition of severe mental and growth retardation known as cretinism. We hypothesize that TH regulation of Dnmt3a plays a pivotal role in normal neurological development, since recent evidence supports that dynamic postnatal regulation of Dnmt3a may be essential for establishing DNA methylation patterns in the developing brain. In the proposed research we will: 1) Investigate the regulation of the Dnmt3a gene in mouse brain in vivo by TH throughout early postnatal development. We will investigate TR recruitment to the Dnmt3a locus, and histone modifications induced by TH. We will directly test whether TH response elements that we identified are required for TH regulation of the Dnmt3a gene. 2) Investigate a role for Dnmt3a in TH-dependent neural cell cycle arrest and differentiation. We will investigate whether cell cycle arrest is mediated by Dnmt3a methylation of the promoter of the cell cycle control gene Cyclin D1, and possibly other E2F target genes. For these experiments we will use mouse neuronal cell models that expresses the 1 isoform of TR. Proper exit from the cell cycle, cell differentiation and the maintenance of cell cycle arrest is essential for normal development. Disruption of these processes in the embryo and fetus can lead to abnormal development; disruption in the adult can lead to cancer and other disease. Thyroid hormone plays a central role in cell cycle exit and cell differentiation in the central nervous system. Several lines of evidence support that Cyclin D1 is directly regulated by Dnmt3a-mediated DNA methylation. We hypothesize that TH regulation of DNA methylation of regulatory regions of cell cycle control genes plays a key role in this process. Successful completion of the proposed research will lead to advances in our understanding of the mechanisms by which DNA methylation is regulated during development, and the roles of TH-dependent DNA methylation in cell proliferation and differentiation.